Our previous studies have shown that the levels of both the acetyl CoA carboxylase and fatty acid synthetase are much lower in the mammary tumors (13762 and R3230AC) as compared to those found in the normal, lactating mammary gland of the rat. Although these results can explain the decreased capacity of these tumors to synthesize long-chain fatty acids from acyl CoA derivatives, the question whether it is the synthesis of these enzymes that is impaired or whether these enzymes are present in a catalytically less active form in these tumors needs resolution. To differentiate between these possibilities, we have concentrated our efforts on acetyl CoA carboxylase, which catalyzes the first committed and rate-limiting step in the biosynthetic sequence of reactions leading to fatty acids. We have purified acetyl CoA carboxylase from the normal lactating mammary gland to a specific activity of 7-8 i.u./mg of protein. This preparation reveals a single protein band when electrophoresed in polyacrylamide gels in the presence of SDS plus 6M urea. Similarly, a single-weight homogeneous species is observed upon sedimentation equilibrium in the presence of 6M guanidine-HCl. Antisera prepared against the purified enzyme have been used to titrate acetyl CoA carboxylase activity purified through identical steps from both the tumors and from the normal gland. The results of these studies show that neutralization of 50% of activity from the tumors requires between 7 to 10 times the amount of antisera needed to achieve the same degree of inactivation with the enzyme of normal gland origin.